Project Summary: Project 1 Cardiovascular disease (CVD), that include increased sensitivity of diabetic (DM) myocardium to ischemic injury, represents the major cause of morbidity and mortality in patients with diabetes. We have uncovered key roles for the receptor for advanced glycation end products (RAGE -gene symbol Ager) in DM and myocardial ischemia/reperfusion (I/R), as global deletion of Ager attenuated myocardial injury, reduced oxidative stress, increased functional recovery and preservation of ATP compared to wild-type (WT) littermates. Mechanisms by which Ager deletion confers metabolic and functional protection in DM and non-DM (NDM) I/R hearts will be the focus of this project. Our program discovered that the RAGE cytoplasmic domain interacts with diaphanous-1 (DIAPH1), an effector of RhoGTPases, is essential for RAGE ligand-mediated cellular migration and activation of cdc42/rac-1. Our recent studies demonstrated that DIAPH1 is expressed in cardiomyocytes (CMs) and that I/R increases expression of Diaph1. Key preliminary data reveal reduced infarct size and improved functional recovery after I/R in (a) WT mice transplanted with bone marrow derived cells from Ager null mice,(b) mice with CM specific deletion of Ager/Diaph1, and (c) WT mice hearts perfused with conditioned media from Ager null or Diaph1 null macrophages (M?s). Novel findings in CMs and M?s reveal that DIAPH1 interacts with mitochondrial GTPase mitofusin2 (MFN2) and augments MFN2's tethering of sarcoplasmic reticulum (SR) to mitochondria (mito) and consequently mito calcium regulation. Importantly, our data shows that high glucose, ligands of RAGE, and RAGE-DIAPH1 interaction further augments DIAPH1-MFN2 driven mito-SR tethering. Taken together, these data led us to hypothesize that RAGE/DIAPH1 mediates DIAPH1-MFN2 driven Mito- SR interactions, altered calcium regulation, cell death signaling and metabolic dysfunction in I/R hearts by cell intrinsic mechanisms in M?s, and via M?-CM cross-talk. We will probe comprehensive mechanisms in cardiac stresses evoked by I/R using murine models, both in the absence and presence of DM. We will employ novel Ager and Diaph1 floxed mice, small molecule antagonists of RAGE-DIAPH1 interaction, state-of-the-art molecular techniques, proteomics, and magnetic resonance spectroscopy to uncover mechanisms of I/R injury in DM hearts.Proposed studies in this project will identify novel mechanism M? specific mechanism, as well as M?-CM cross talk mechanisms by which RAGE-DIAPH1 modulates I/R injury in hearts, particularly in DM hearts. Identification of these mechanisms, along with testing of novel small molecules that block RAGE/DIAPH1 interaction, will pave the way for therapeutic interventions to protect DM hearts from I/R injury. Project 1 will work closely with Projects 2 and 3 and the two Cores to achieve these goals.